The invention relates to cytotoxic agents and radiotherapy effective against hypoxic cells. More specifically, the invention relates to certain novel 1,2,4-benzotriazine oxides, to methods of selectively killing tumor cells and/or sensitizing tumor cells to radiation using selected 1,2,4-benzotriazine oxides, and to novel synthetic methods.
Hypoxic cell radiosensitizers are compounds that selectively increase the sensitivity of hypoxic cells to destructive radiation. Cytotoxins which have enhanced activity under hypoxic conditions also provide a means for selective destruction of cells under low oxygen pressure. This specificity for hypoxic cells is important because it is tumors that are typically characterized by such cells. Virtually all tumors which are present as solid masses contain these cells, while normal cells generally have an adequate supply of oxygen. Accordingly, anti-tumor agents can be made selective for tumors by virtue of high activity under hypoxic conditions, and radiation can be employed more effectively in the presence of these sensitizers.
Of course, the use of radiation treatment to destroy tumor cells is only practical if damage to the surrounding normal tissue can be minimized or avoided. The effects of radiation are enhanced by the presence of oxygen, and it is established that as the dose of radiation is increased, the effectiveness of the radiation in destroying target cells is enhanced most dramatically when oxygen is present. Therefore, selectivity for tumor cells toward radiation is difficult to achievexe2x80x94normal cells, in view of their oxygen supply, are generally more susceptible to radiation than the target tumor cells. It is therefore desirable to provide a means of sensitizing tumor cells, but not the surrounding tissue, to radiation treatment. One solution would be to increase the supply of oxygen to these tumor cells. This, however, has proved difficult to do.
Various heterocyclic compounds and in particular those with oxidized nitrogen moieties, have been used to radiosensitize hypoxic tumor cells. Indeed, it has been postulated that the oxidized nitrogen. functionality is responsible for this activity. Nitroimidazoles, particularly misonidazole (MIS) and metronidazole have been studied extensively, and MIS is commonly used as a standard in in vitro and in vivo tests for radiosensitizing activity. (See, e.g., Asquith, et al, Radiation Res (1974) 60:108-118; Hall, et al. Brit J Cancer (1978) 37: 567-569; Brown, et al, Radiation Res (1980) 82:171-190; and U.S. Pat. No. 4,371,540. The radiosensitizing activities of certain 1-substituted 3(5)-nitro-s-triazoles and of various quinoxaline-1,4-dioxide derivatives have also been disclosed.
In addition, U.S. Ser. No. 730,761, filed May 3, 1985, and U.S. Ser. No. 788,762, filed Oct. 18, 1985 assigned to the same assignee and incorporated by reference disclose a group of radiosensitizers that do not contain oxidized nitrogenxe2x80x94the substituted benzamides and nicotinamides and their thio analogs. These compounds, nevertheless, are radiosensitizers. It is important to distinguish the ability to sensitize hypoxic cells selectively, for instance, by enhancing their oxygen supply, from another mechanism commonly encountered for xe2x80x9csensitizingxe2x80x9d cells: inhibition of the enzyme poly(ADP-ribose)polymerase, which is believed to be essential in the repair of irradiated cells after radiation. This repair mechanism is operative in both hypoxic tumor cells and in normal cells. Hence, administration of xe2x80x9cradiosensitizersxe2x80x9d which operate according to this latter mechanism does not accomplish the desired purpose of selectively sensitizing the target tumor cells.
A group of compounds which has not previously been suggested for use in either selectively killing hypoxic cells or in radiosensitizing such cells is 3-amino-1,2,4-benzotriazine 1,4-di-N-oxide and related compounds. Related U.S. Pat. Nos. 3,980,779; 3,868,371; and 4,001,410 disclose the preparation of a group of these compounds and their use as anti-microbial agents, particularly by addition of these materials to livestock fodder. U.S. Pat. Nos. 3,991,189 and 3,957,799 disclose derivatives of these compounds bearing substituents on the nitrogen of the 3-amino group. These compounds also have anti-microbial activity.
The present invention provides additional compounds which specifically radiosensitize hypoxic cells and which, furthermore, are directly cytotoxic to hypoxic cells both in vitro and in vivo. Therefore, administration of these compounds prior to or following radiation treatment of tumors selectively kills the hypoxic (tumor) cells which survive the radiation dose. Both the ability of these compounds to radiosensitize hypoxic cells and especially their ability to selectively kill hypoxic cells directly are unexpected properties of these compounds.
The invention also provides novel 1,2,4-benzotriazine oxides useful as radiosensitizers and/or selective cytotoxic agents; methods of synthesizing the compounds; and methods of administering the compounds to achieve radiosensitization and/or selective cell killing.
The invention provides a valuable addition to the group of compounds currently available as selective radiosensitizers and selective cytotoxic agents for hypoxic tumor cells. Some of the compounds now newly shown to be useful in this regard are known compounds. Others are themselves novel.
Accordingly, one aspect of the invention is a method of radiosensitizing hypoxic tumor cells by administering to these cells a compound of the formula: 
wherein X is H; hydrocarbyl (1-4C); hydrocarbyl (1-4C) substituted with OH, NH2, NHR or NRR; halogen; OH; alkoxy (1-4C); NH2; NHR or NRR; wherein the various R groups are independently selected from lower alkyl (1-4C) and lower acyl (1-4C) and the R""s may themselves be substituted with OH, NH2, alkyl (1-4C) secondary and dialkyl (1-4C) tertiary amino groups, alkoxy (1-4C) or halogen. In the case of NRR, the two R""s can be linked together directly or through a bridge oxygen into a morpholino ring, pyrrolidino ring or piperidino ring;
n is 0 or 1; and
Y1 and Y2 are independently either H; nitro; halogen; hydrocarbyl (1-14C) including cyclic and unsaturated hydrocarbyl, optionally substituted with 1 or 2 substituents selected from the group consisting of halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), primary amino (NH2), alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, dialkyl (1-4C) tertiary amino where the two alkyls are linked together to produce a morpholino, pyrrolidino or piperidino, acyloxy (1-4C), acylamido (1-4C) and thio analogs thereof, acetylaminoalkyl (1-4C), carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C), alkylsulfonyl (1-4C) or alkylphosphonyl (1-4C), wherein the hydrocarbyl can optionally be interrupted by a single ether (xe2x80x94Oxe2x80x94) linkage; or wherein Y1 and Y2 are independently either morpholino, pyrrolidino, piperidino, NH2, NHRxe2x80x2, NRxe2x80x2Rxe2x80x2 O(CO)Rxe2x80x2, NH(CO)Rxe2x80x2, O(SO)Rxe2x80x2, or O(PORxe2x80x2)Rxe2x80x2 in which Rxe2x80x2 is a hydrocarbyl (1-4C) which may be substituted with OH, NH2, alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino, pyrrolidino, piperidino, alkoxy (1-4C), or halogen substitutents, or pharmacologically acceptable salts of said compound.
In another aspect, the invention provides an improved method of fractionated radiotherapy which involves treating the cells requiring radiotherapy with a 1,2,4-benzotriazine oxide of Formula (I), as just defined, before or after subjecting the treated cells to a plurality of distinct radiation doses over an extended period of time, each of the radiation doses being less than about 5 Gy.
The compounds useful in conjunction with t presently disclosed radiosensitizing methods, therefore, are the mono- or dioxides of optionally substituted 1,2,4-benzotriazine which may contain a hydrocarbyl (1-4C), hydroxyl, alkoxy or amino group, either substituted or unsubstituted, in the 3-position, and their pharmacologically acceptable salts as set forth in Formula I
The invention also provides a method for selectively killing hypoxic tumor cells using certain of these 1,2,4-benzotriazine oxides. The compounds which are useful as selective cytotoxic agents are a subset of the above-defined compounds useful as radiosensitizers. That is, while all of the compounds defined by Formula (I) are generally effective as radiosensitizers, only those compounds unsubstituted at the 3-position or having a 3-amino or 3-hydrocarbyl (1-4C) substituent (i.e., Xxe2x95x90H, hydrocarbyl (1-4C), NH2, NHR or NRR with each R as defined above) and which are di-N-oxides (n=1) are effective cytotoxic agents. In this aspect, the invention provides a method of selectively killing hypoxic tumor cells by administering one or more of these compounds (or its salts) to the hypoxic tumor cells.
Certain of the compounds encompassed by Formula (I) are already known in the art as being useful for other purposes; other compounds are novel. The novel compounds encompassed by the present invention and which may be prepared by methods disclosed herein include compounds represented by Formula (I), in which the substituents fall into the following three classes:
I. X is OH, alkoxy (1-4C), NHR or NRR where each R is independently an alkyl of 1-4 carbon atoms, or acyl of 1-4 carbon atoms, or where the two R groups are alkyls linked together to form a pyrrolidino or piperidino ring or linked through an oxygen to form a morpholino ring, and the R groups may be further substituted with OH, NH2, alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, pyrrolidino, piperidino, alkoxy (1-4C), or halogen substitutents;
n is 1; and
Y1 and Y2 are independently either H; nitro, halogen; hydrocarbyl (1-14C) including cyclic and unsaturated hydrocarbyl, optionally substituted with 1 or 2 substituents selected from the group consisting of halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), primary amino (NH2), alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, dialkyl tertiary amino where the two alkyls are linked together to produce a morpholino, pyrrolidino or piperidino, acyloxy (1-4C), acylamido (1-4C) and thio analogs thereof, acetylaminoalkyl (1-4C), carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C), alkylsulfonyl (1-4C) or alkylphosphonyl (1-4C), wherein the hydrocarbyl can optionally be interrupted by a single ether (xe2x80x94Oxe2x80x94) linkage; or wherein Y1 and Y2 are independently either morpholino, pyrrolidino, piperidino, NH2, NHRxe2x80x2, NRxe2x80x2Rxe2x80x2 O(CO)Rxe2x80x2, NH(CO)Rxe2x80x2, O(SO)Rxe2x80x2, or O(PORxe2x80x2)Rxe2x80x2 in which Rxe2x80x2 is a hydrocarbyl (1-4C) which may be substituted with OH, NH2, alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino, pyrrolidino, piperidino, alkoxy (1-4C), or halogen substitutents. Pharmacologically acceptable salts of these compounds are also included in this class of compounds.
II. X is NH2;
n is 1; and
Y1 and Y2 are chosen such that one but not both may be hydrogen and one or both may independently be either nitro, saturated or unsaturated hydrocarbyl of 7-14C, or unsaturated hydrocarbyl of 2-6C, optionally substituted with 1 or 2 substituents selected from the group consisting of halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), primary amino (NH2), alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, dialkyl tertiary amino where the two alkyls are linked together to produce a morpholino, pyrrolidino or piperidino, acyloxy (1-4C), acylamido (1-4C) and thio analogs thereof, acetylaminoalkyl (1-4C), carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C), alkylsulfonyl (1-4C) and alkylphosphonyl (1-4C), wherein the hydrocarbyl can optionally be interrupted by a single ether (xe2x80x94Oxe2x80x94) linkage; or wherein Y1 and Y2 are independently either morpholino, pyrrolidino, piperidino, NH2, NHRxe2x80x2, NRxe2x80x2Rxe2x80x2 O(CO)Rxe2x80x2, NH(CO)Rxe2x80x2, O(SO)Rxe2x80x2, or O(PORxe2x80x2)Rxe2x80x2 in which Rxe2x80x2 is a hydrocarbyl (1-4C) which may be substituted with OH, NH2, alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino, pyrrolidino, piperidino, alkoxy (1-4C), or halogen. substitutents. Pharmacologically acceptable salts of these compounds are also included in this class of compounds.
III. X is hydrogen or hydrocarbyl (2-4C) optionally substituted with OH, NH2, alkoxy (1-4C) or halogen substituents;
n is 1; and
Y1 and Y2 are independently either H; nitro; halogen; hydrocarbyl (1-14C) including cyclic and unsaturated-hydrocarbyl, optionally substituted with 1 or 2 substituents selected from the group consisting of halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), primary amino (NH2), alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, dialkyl tertiary amino where the two alkyls are linked together to produce a morpholino, pyrrolidino or piperidino, acyloxy (1-4C), acylamido (1-4C) and thio analogs thereof, acetylaminoalkyl (1-4C), carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C), alkylsulfonyl (1-4C) or alkylphosphonyl (1-4C), wherein the hydrocarbyl can optionally be interrupted by a single ether (xe2x80x94Oxe2x80x94) linkage; or wherein Y1 and Y2 are independently either morpholino, pyrrolidino, piperidino, NH2, NHRxe2x80x2, NRxe2x80x2Rxe2x80x2 O(CO)Rxe2x80x2, NH(CO)Rxe2x80x2, O(SO)Rxe2x80x2, or O(PORxe2x80x2)Rxe2x80x2 in which Rxe2x80x2 is a hydrocarbyl (1-4C) which may be substituted with OH, NH2, alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino, pyrrolidino, piperidino, alkoxy (1-4C), or halogen substitutents. Pharmacologically acceptable salts of these compounds are also included in this class of compounds.
The invention also provides a straightforward, one-step synthesis for preparing 1,2,4-benzotriazine oxides unsubstituted at the 3-position (i.e., the compounds of Formula (I) wherein Xxe2x95x90H) by treating the corresponding 3-amino-1,2,4-benzotriazine oxide with a lower alkyl nitrite under reductive deaminating conditions.